This proposal describes procedures designed to elucidate the differing roles played by pteridines and folates in selected biological systems. Experimental approaches will include the use of photolabile probes to examine the nature of the nucleotide or substrate binding sites of rat liver dihydrofolate reductase, dihydropteridine reductase, and phenylalanine hydroxylases, Lactobacillus casei thymidylate synthetase, and bovine adrenal medulla tyrosine hydroxylase. In addition, more extensive comparison of the structural properties of the rat liver dihydropteridine and dihydrofolate reductases will be made to attempt to identify the differing requirements of the "quinonoid" and 7,8-dihydro-pteridien or -folate binding sites. The oxidative pathways of substituted and unsubstituted tetrayhydropteridines will be examined as aids to understanding the enzymatic mechanisms of the tetrahydropteridine-requiring monooxygenases, and a series of fluorescent methotrexate derivatives will be synthesized as probes for dihydrofolate reductases and folate transport proteins from different celllines to amplify the results already obtained with fluorescein-diaminopentyl-methotrexate. In order to expand our interests in the concept of carrier-mediated drug delivery, methotrexate will be bound to poly-L-lysine and L1210 cell-specific monoclonal antibodoes, and emphasis will be placed on determining how carrier structure and cell membrane-carrier interaction influence effective chemotherapy.